Fluorometric Analysis - ACS Publications

1432 (August 1945). Organic reagents foruranium analysis. (366) Webb, J. A. V., S. African Ind. Chem., 4, 189-91 (1950). Rapid sodium diethyldithiocar...
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V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 mination of sulfur by Grate method. Photometric detection of titrimetric end point. (365) Ware. E., U. S. Atomic Energy Commission, Rept. MDDC1432 (August 1945). Organicreagents for uranium analysis. (366) Webb, J. A. V., S. A f r i c a n I n d . Chem., 4, 189-91 (1950). Rapid sodium diethyldithiocarbamate method for determination of copper in straight carbon steels. (367) Wenger, P. E., Mikrochemie ver. Mikrochim. A c t a , 36/37, 94104 (1951). Role of microchemistry in new fields of analytical chemistry. (368) Test, P. W., ANAL.CHEM.,23, 51-9 (1951). Inorganic microchemistry. (369) Ibid., pp. 176-80. Microchemical applications of catalytic and induced reactions. (370) West, P. W., and Carlton, J. K., Ibid., 22, 1055-6 (1950). Specific spot test for gold, employing pararosaniline hydrochloride. (371) West, P. W.,and Conrad, L. J., Ibid., 22, 1336-7 (1950). Spot-test detection of antimony by means of gossypol. (372) West, P. W.,and Conrad, L. J., A n a l . Chin. A c t a , 4, 561-5 (1950). Comparison of coprecipitation of cations by organic and inorganic precipitants. (373) West, P. IT., and Conrad, L. J., Mikrochemie zer. Mikrochim. A c t a , 35, 443-8 (1950). Detection of vanadium by spot tests. (374) West, P. K., and De Vries, C. G., ANAL.C m x , 23, 334-7 (1951). Nature of cobalt thiocyanate reaction. (375) Rest, P. W., and Granatelli, Lawrence, Mikrorhewiie ver. M i k r o c h i m . A c t a , 38, 63-5 (1951). Microscopic detection of chromium as chromium(II1). (376) West, P. TV., and Hamilton, IT. C., Ibid., 38, 100-13 (1951). Effect of media upon spot-test reactions. (377) West, T. S.,Metallurgia, 43, 41-6 (1951). Separation methods in metallurgical analysis. (378) Whittles, C. L., and Little, R. C., J . S c i . Food. A g . , 1, 323-6 (1950). Colorimetric determination of potassium in soil extracts. (379) Rilberg, E., 2.anal. Chem., 131, 405-9 (1950). Determination of lithium by flame photometry.

85 (380) Willard, H. H.. and Horton, C. A., ANAL.CHEM,22, 11904 (1950). Indicators for titration of fluoride with thorium. (381) Ibid., pp. 1194-7. Photofluorometric titration of fluoride. (382) Willard, H. H., and Sheldon, J. L., I b i d . , 22, 1162-6 (1950). Separation of iron as basic formate from homogeneous solution with urea. (383) Williams, H. A., A n a l y s t , 75, 510-21 (1950). Determination of fluoride by etching. (384) Willson, A. E., ANAL.CHEJI., 22, 1571-2 (19!0). Volumetric determination of calcium and magnesium in leaf tissue. (385) Wilson, D. IT., Roy. I n s t . Chem. Lectures, Monographs Repts., 4, 5-15 (1950). Maintenance and precision of microchemical balance. (386) Wright, P. IT., J . SOC. Chem. Ind., London, Suppl. Issue, 2, S69-S70 (1950). Determination of copper by dithio-oxamide in lead and lead alloys. (387) Yao, Yu-lin, and Yu, Pe-nien, Science Record, 2, 377-80 (1949). Colorimetric determination of traces of arsenic in tin, (388) Yoshino, Y., and Kojima, M., B u l l . Chem. SOC.J a p a n , 23, 46-7 (1950). Separation of small amount of titanium from iron by ion exchange resin. (389) Young, R. S., A n a l y s t , 76, 49-52 (1951). Determination of gold, palladium, and platinum by dithiaone. (390) Young, R. S., Leibowitz, A., I r o n A g e , 164, 75-6 (November 1949); J. I r o n Steel Inst., 165, 363 (1950). Molybdenum separation in iron alloys. (391) Zacherl, hl. K., Mzlt. chem. Forsch.-Inst. I n d . osterr., 4, 27-9 (1950). Present situation of Austria in microchemistry(392) Zettemoyer, A. C., and Walker, W.iC., Am. I n k M a k e r , 28, No IO, 69-71 (1950). Cobalt analysis in inks and driers. (393) Zhivopistsev, V. P., Doklady A k a d . N a u k S.S.S.R., 73, 1193-6 (1950). Possible applications of diantipyrylmethane in inorganic analysis. (394) Ziliani, Giuseppe, M e t . ital., 42, 2 2 5 9 (1950). Microscopic method for determination of oxygen in steels. RECEIVED November 8, 1 9 6 1

FLUOROMETRIC ANALYSIS CHARLES E. WHITE, University of Maryland, College Park, M d .

T

HIS revieLT on fluorometric analysis covers the 2-year period from approximately October 1949 to October 1951, and is a continuation of previous surveys (170, 171). BOOKS AND GENERAL ARTICLES

Several books have been published during this period which are of interest from a general standpoint. Under the title, “Lumineszenz,” Bandow ( 7 ) has compiled a book on the applications of fluorescence in physics, chemistry. and biology. The chief value of this book to the analyst is in the review of German literature. Strugger’s (166) treatise on the use of fluorescence microscopy in microbiology gives the general techniques, names many of the fluorescent dyes used, shows the effect of p H , and lists 102 references. Leverenz (98)gives an excellent treatment of the theory of fluorescence and phosphorescence of solids, scintillation counters, and the composition of phosphors; 750 references are included. Kroger’s (93) book, which is also concerned with the fluorescence of solids, includes tables giving the fluorescence of many inorganic compounds. Curie ( 2 7 ) in a book on the general topic of fluorescence and phosphorescence discusses the general theory of the production of luminescence in organic and metallo-organic complexes and the relation of fluorescence to the constitution of the molecule. A chapter on cathode luminescence, triboluminescence, chemiluminescence, etc., is also included. Foster ( 5 2 ) has published a book on fluorescence of organic compounds which does not give analytical applications, b u t is of interest from a theoretical standpoint. Sandell (138)in the second edition of his text gives fluorometric methods for aluminum, beryllium, gallium, indium, scandium, thallium, uranium, zinc, and rare earths. H e also includes a theoretical discussion on the application of the

Beer-Lambert law to fluorescence in the same vein as the excellent dcvelopment of this topic by Lothian (101) and Kavanagh (84). Several general articles on fluorometric analysis have appeared. DBribBrB (34) reviews recent applications of ultraviolet light in analysis; Radley (131) discusses fluorescence methods in the food industry; White (178) gives specific procedures and apparatus for use in the usual scheme of qualitative analysis; and Levskin (100)reviews the advances in luminescence analysis with 51 references. APPARATUS

Sources of Ultraviolet Radiation. A new series of lamps for both the longer ultraviolet light a t 3650 A. and the shorter a t 2537 A. has been announced by Ultra-Violet Products, Inc. (162). These lamps are of convenient laboratory type and are equipped with mercury vapor lamps and 360 B. L. tubes. Several firms (154, 162) now market fixtures for 4-watt fluorescent lamps which may be used with the 360 B. L. tubes for qualitative analysis. As these fixtures are made for daylight lamps, the reflectors are usually white enamel and should be replaced by aluminum reflectors for ultraviolet lamps. T h e Menlo Research Laboratory (108) sells an instrument known as a daylight fluorescence tester u hich includes a viewing apparatus and a source of ultraviolet radiation activated by a battery or ordinary poner supply. Bn interchangeable head converts the source to either 3650 or 2537 A. Fluorometers. A new fluorometer has been widely advertised by the Central Scientific Co. (24). This instrument is equipped with two phototubes, so that measurement may be made on

86 fluorescent solutions over the entire visible spectrum. Several instrument makers (5, 128, 161) have developed moderately priced line-operated electron Inultiplier photometers which are equipped with a unit that is easily adapted to the measurement of fluorescent light. Directions for making and improving fluorometers have been given by many authors along with discussions of particular methods. Alford and Daniel (1) have devised an especially stable, sensitive, and flexible instrument using a VX-41A electrometer tube as an amplifier in a DuBridge-Brown type circuit. Kaufman (83)and his associates have improved the Argonne Model XI fluorometer and use an AH6 lamp to increase sensitivity. Fletcher and May (@) have built a transmission fluorometer for use in the determination of uranium in fluoride melts. I n this instrument, the sample is placed between the ultraviolet source and the photocell. This makes a simple arrangement of the optical parts and is about ten times as sensitive as a reflection-type instrument with the same amplifying unit. Hilger and Watt in England have designed an instrument for the measurement of the fluorescence of solids, in which the optical parts are all on a horizontal axis. I n this apparatus the ultraviolet rays from a mercury vapor lamp pass through a condenser and are reflected by a mirror to the sample. Another mirror picks up the fluorescent light and transmits it through a lens system to a photoelectric multiplier tube. Davey and Florida (89) have given this fluorometer extensive tests and conclude that the workmanship is excellent. They suggest that the turntable holding the sample be changed to a slide in order to facilitate the operation of this unit. Wheelock (169) describes a fluorometer designed with a water cooling jacket around the mercury vapor lamp. Wokes and Slaughter (179)have increased the sensitivity of the Spekker instrument 75-fold by replacing the barrier layer cells with a photomultiplier tube. The general use of electron multiplier phototubes in the measurement of weak light intensities is discussed by Oldenberg and Broida ( 141 ) and a balanced circuit type of instrument is described. Stephen (163’) uses an instrument for the determination of thiamine by which 750 galvanometer units are registered with 1 microgram of thiamine in 10 ml. of solution. A comparison fluorometer of high sensitivity is described by Larsen (95), in which an automatic device switches the light from the known to the unknown sample a t 60 cycles per second. Pllinard and Eicher (110) have devised a two-channel fluorometer for in vivo studies to measure the time of appearance and rate of change of fluorescein on tissue areas after injection. At the Pittsburgh spectroscopy conference, Priestley ( 129) described a modification of the Beckman spectrophotometer in which he used a 1P28 electron multiplier tube with a Brown recorder to determine the fluorescent spectra of polycyclic hydrocarbons. *in apparatus to facilitate the excitation and observation of chromatographic columns, developed by an instrument company (187), consists chiefly of an arrangement whereby a mercury vapor lamp is easily passed over the column. Brumberg (21) has devised an apparatus Jvhich he calls an ultrachemiscope for examining paper chromatograms. In this instrument the ultraviolet light passes through the paper and strikes a fluorescent screen. This screen is made of materials that are excited by different wave lengths of energy and produce different colors; hence the absorption pattern of the paper is easily observed. Brockman and Beyer (19) use photographic techniques for recording the fluorescent zones on chromatographic columns. Harvalik (7’2) has employed an electronic image converter for studying the fluorescence in chromatography when the exciting rays extend from the ultraviolet to the infrared. Recording spectroradiometers have been developed by Parsons and his associates ( I H ) for the automatic recording of the energy distributions emitted by luminescent materials. The fluorescent spectra of dyestuffs on cloth have been obtained with modifications of the Carey (109) and Beckman (143) spectrophotometers

ANALYTICAL CHEMISTRY Vendt (164) claims that an excellent condenser and filter for the excitation rays in the 3650 A. range can be made by filling a 40- or 60-watt clear electric light bulb a i t h 10% cobalt sulfate solution. The use of a solution of tetrammine cupric sulfate as a filter in fluorescence microscopy is described by Zanikov (181). Quinine sulfate has long been used as a standard in setting instruments. Bird (12) h d s that this compound is adsorbed on the walls of test tubes after long standing. Treatment of the tubes with hot alkali renders them nonadsorptive. INORGANIC APPLICATIONS

Aluminum. For some years the fluorescence of the 8-quinolinol complexes of gallium and indium dissolved in chloroform has been used in the determination of these elements. It has now been shown that this same method may be employed for the analyses of aluminum. Tullo (160) and his associates have applied this method for the determination of aluminum in beer; and Grimaldi and Levine (64)use it for the analysis of aluminum in phosphate rock. I n both cases the method is rapid and accurate for even extremely small quantities. In rock analysis a great advantage lies in the fact that small original samples can be used. Wiberley and Bassett (174) describe the procedure where aluminum oxinate is dissolved in chloroform as a colorimetric method for determining aluminum in steel; in another article ( 9 ) they indicate that the fluorescence of the chloroform extract may also be used as a measure of the aluminum concentration. Kulberg and Mustafin (94) report that certain of the chlorohydroxyanthraquinones form fluorescent coniplexes with aluminum which are not destroyed by concentrated hydrochloric acid. If this fluorescence is sufficiently intense for analytical purposes, the reaction may be very useful, as the other fluorometric reagents are ineffective in even dilute hydrochloric acid. Rouir and Deita (136) have found the morin method useful for determining quantities of aluminum in the range of 0.001% in bronzes and brasses. Bishop (16) has made an extensive study of the effect of anions on the morin test for aluminum, gallium, beryllium, and zinc, and has shown that the aluminum-morin complex may Ire used as a sensitive test for citrate, fluoride, oxalate, phosphate, tartrate, and vanadate ions. A 0.2% solution of morin in 0.01 S sodium hydroxide is recommended for qualitative work; the alcohol solution is more seneitive and should be used when quantitative results are desired. Morin is used by Klemperer and Martin (90) as the reagent for the determination of beryllium in biological materials. The smltllest amount detected by this method was 0.05 microgram. The applications of 8-quinolinol (oxine, 8-hydroxyquinoline) as a reagent in fluorometric analqses are numerous. Gentry and Sherrington (58) have determined the pH conditions for the complete extraction with chloroform of the oxinates of molybdenum, iron, tin, copper, nickel, aluminum, and manganese. They show that this is a useful method for the separation of these metals and state that the chloroform solution may be used for the fluorometric determinationas well as for thedirect colorimetric analyses. Feigl and Heisig (46) have studied the detection of calcium, magnesium, and aluminum on paper with 8-quinolinol as the reagent. Aluminum is the only one of these in which the fluorescence will persist in the presence of acetic acid vapor. i f the paper is exposed to fluoride vapor, the aluminum 8-quinolinol diminishes in fluorescence and a test for fluoride results. A test for cyanide ion is based on the liberation of 8-quinolinol from the copper complex and the determination of the 8-quinolinol with aluminum. Beryllium may be separated from aluminum qualitatively with paper chromatography and both identified by spraying with 8quinolinol (182). The beryllium moves considerably faster than aluminum if a solvent of 80% butanol and 20Yo concentrated hydrochloric acid is used, An extensive study of the analytical applications of the 8-quinolinol derivatives of gallium and thallium has been made by Moeller and Cohen (112) and their article

V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 should be read by anyone using the chloroform extraction of oxinatsa. Dudley (36) has shown that t h e gallium oxinate-chloroform method may be used in the determination of gallium in animal tissue. White and associates (173) have used the lithium-8-quinolinol complex for the determination of lithium in rocks. This serves as one of t h e simplest methods of comparing t h e lithium content of reagent5 to a standard. The fluorometric method for the determination of uranium, which uses the sodium fluoride melt, has been reviewed in several publications (44, 63, 116, 130, 134). Grimaldi and his associates (66) have applied this method to the determination of uranium in shale and phosphate rock for field a8 well aa laboratory use. Willard and Horton (1‘76) have studied a number of the colorimetric and fluorometric indicators for the titration of thorium with fluoride. Of the 200 compounds used 162 showed no change in either the visible or ultraviolet light, and among these morin and quercetin were the best fluorescence indicators, With these latter indicators, the results of the fluoride titration (176) were better than those n i t h alizarin red S when the fluoride was in amounts greater than 2 mg. Alford and others ( 2 ) have introduced flavonol as a new fluorometric reagent for zirconium. The fluorescence of this complex is very intense and it persists in 0.2 N sulfuric acid solution. Hafnium is the only other element that gives a fluorescence with flavonol in 0.2 M sulfuric acid. The method greatly simplifies the determination of zirconium in ores. I n a paper concerned with the colorimetric determination of boron with 1,l-dianthrimide, Ellis and his associates (40) show that I-amino4hydroxyanthraquinone may be used for thequantitative determination of this element by measuring the fluorescence in concentrated sulfuric acid solution. They prefer the 1,l-dianthrimide reagent. In an article on the use of ultraviolet light in the food industry, Radley (181) suggests that fluorometric methods be used for boron, sulfur dioxide, and reducing agents. -4rather unusual use of fluorescence in analysis has been described by Brumberg and others (.%), who utilize t h e absorption characteristics of various compounds to indicate t h e end point in titrations. Filtered ultraviolet light is passed through the solution being titrated and permitted to come in contact with a fluorescent screen. The end point is observed by the appearance or extinction of the fluorescence. A qualitative test for zinc and cadmium is indicated by DenigBs (83) as a result of a study of the reaction of resorcinol with phthalic anhydride. Resorcinol in concentrated hydrochloric acid with zinc gives a green fluorescence and with cadmium produces a violet-rose colored solution which does not fluoresce. The application of fluorescence microscopy in mineralogy has been reviewed by Haberlandt (71) and 79 references are given. This same author has determined the fluorescent spectra of several minerals (‘70) and has made an extensive study of the rare earths in fluorite (69). The fluorescent test for some of the rare earths can be extremely sensitive-for example, 0.001 microgram of yttrium can be easily detected (119). Siobium and uranium (106) in columbite can be detected through the sodium fluoride fusion technique, Wilson (177) has studied the fluorescence of feldspars with short-wave-length ultraviolet light and indicates that this is a useful property for distinguishing feldspar from other minerals. Dement (32) and Hershey (74) have published t rtbles showing the fluorescence of many minerals. It is a question whether the x-ray fluorescence method of analysis belongs in this review or under spectrographic methods. I n this technique t h e exciting ray and the emitted rays are both in the x-ray region and the emission is detected with a Geiger tube. Solid specimens are used for analysis and are not altered or destroyed. Birks and his associates have applied this method to the determination of lead and bromine in ethyl fluid (16) and to the analysis of mixtures of hafnium, zirconium, columbium, tantalum ( f a ) , and uranium (14).

87 ORGANIC APPLICATIONS

Acetol may be determined fluorometrically by reaction with o-aminobenzaldehyde to form bhydroxyquinaldine (61). This is sensitive to 0.3 mg. of acetol per liter. Thornton and Speck (168) have devised a procedure for the determination of pyruvaldehyde which is based on its reaction with chromotropic acid. T h e product has a n intense green fluorescence which gives a straight-line relationship for concentrations of pyruvaldehyde from 0.0 to 1.5 mg. per liter. A method designed for the analysis of 2-nitronaphthalene in the presence of 1-nitronaphthalene depends on the sulfonation and reduction of these compounds. Filters are used to exclude the dull green fluorescence of t h e alpha isomer and the intensity of the blue fluorescence of the beta isomer is measured The procedure detects 0.05% of the beta isomer (43). Benzil may be determined by its condensation product mith m-diethylaminophenol (141). This reaction forms a blue-red dye with a yellow-red fluorescence. Benzil concentration up to 0.03 mg. per ml. gives a straight line Rhen plotted against galvanometer readings. Alkylated aminophenols can be determined by the condensation reaction with diketones. A qualitative fluorescence test for malic acid has now been applied quantitatively by Leininger and Katz (9’7). This involves the reaction of malic acid with 2-naphthol in the presence of concentrated sulfuric acid to produce a very highly fluoresceni benzocoumarin. Hummel (78) uses orcinol as the reagent for the determination of malic acid. The methyl coumarin produced has a bright blue fluorescence. Alloxan monohydrate may be determined as riboflavin when condensed with ~-l-ribitylamino-2-amino-4,5-dimethylbenzene hydrochloride (159). A qualitative test for acenaphthene is achieved by nitrating this compound and then boiling it with lead in the presence of glacial acetic acid (180). This reaction producee a compound which has a green fluorescence and is specific for acenaphthene. Radley (131) uses acenaphthene 5-carbolic acid to test for formaldehyde; these two compounds react in sulfuric acid to produce , a greenish yellow fluorescence. Under t h e same conditions tartaric acid produces a yellow blue, glycerol a yellow, and ethylene glycol a golden yellow fluorescence. Anthrone (132) also serves as a reagent for t h e latter three substances. The variation of the intensity of fluorescence with pH gives a characteristic curve, which in some cases can be used for identification purposes. Curves of this type have been found to be similar for flavin compounds and these curvea differed from those of the 4-pteridine series (86). The color and relative intensity of fluorescence of 98 coumarin derivatives over a pH range of 1.6 to 12.6 have been reported (61). The influence of the position of the hydroxyl, carboxyl, and methyl groups is definite. The use of these p H curves in identification of compounds is shown by data on scopoletin, which gives a pH-fluorescence intensity curve similar to t h r coumarin from avena roots. Sisteen synthetic coumarins were tested for change in fluorescence over the range of pH 3 to 10, and seven shoxed marked color change near the neutral point (118, 148). Thc presence of chlorophyll in living chloroplasts may be observed b y fluorescence microscopy. When the temperature on the cell structure is suddenly increased, the red fluorescence of the entire chloroplast shows a great increase in intensity (69). This increase in fluorescence, observed under the microscope, presents a simple means of making a submicroscopic alteration evident which may not be seen by the ordinary microscopi=. Phthalic acid esters may be determined by treating them with sulfuric acid to form phthalic anhydride, which is in turn fused with resorcinol to prcduce fluorescein (163). Gage and Wender (66) have shown that binary mixtures of flavonol-3-glycosides can be separated by paper partition chromatography and the individual pigment zones located with ultraviolet light. The fluorescences of acridine and many of its derivatives have

ANALYTICAL CHEMISTRY

88 been measured by Villemey (166). For low concentrations the intensity of the fluorcscence was found to be proportional to the concentration. The fluorescent spectra of the acridine derivatives were also determined. The fluorescence spect,ra of several polycyclic aromatic compounds have been determined by Scott (145, 147). The usual correlation between the corresponding absorption and fluorescence spectra was found and the effect of the substituent groups was determined. The use of fluorescence as an analytical tool does not seem promising for the study of high polymers (157). Conrad ($6) has shown that the aromatic hydrocarbons in gasoline can be identified by a combination of chromatographic and absorption t,echniques. Parasheen, a highly fluorescent substance soluble in hydrocarbons, is added before adsorption of mixture on a silica column. Fluorescent methods are reported to hold promise for geophysical prospecting and soil analysis for petroleum (45, 151). The chromatographic fraction of black oils is described by Lawrence and Barby (96) and the dispersion, color, and fluorescence data for various fractions are recorded. Carcinogcnic substances, chiefly 3,4-benzopyrene, in tars and resiris have been determined by the fluorescence of the paraffin oil gram extract of these substrances (79). By this method 5 X of 3,4-benzopyrene per gram of tar is easily detected. Fluorescence offerfi R means of distinguishing trans-t,rans from cis-trans configurations in thc 1,4diarylbutyldienes (75). BIOLOGICAL APPLICATIONS

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In a review of the use of ultraviolet radiation in the food industry, Radley (131) discusses the examination of oils, fats, milk, etc., and describes the tests for organic acids, sugars, glycerol, and preservatives. Bergholts (21) has assembled a review with 90 references on the use of luminescence analysis of some biologically active substances in animal organisms. The determination of vitamins B, and B? continues to he the subject of consider~hleresearch. Getirner (63) in Germany and the Association of Vita,min Chcmists (6)in this country havepublished books which include t,he recommended methods for vitamin assay. A report from the Subcommittee on Vitamin EEtimations of the British Society of Public Analysts gives an excellent review of the fluorometric method for B, (SO). Ridyard (133) has made a n analysis of the factors affecting the recovery of B1 and presents a theoretical discussion of the fluorometric estimation from the standpoint of Beer's law. Stack (1.52) recommends t8hat a solution of 0.0001 gram of quinine per 100 mi. of 0.1 K sulfuric acid be used as a standard for thiamine determinations. Thiamine determination in wheat flour and bread is given by Petit (186). The determination of thamine in urine has been dealt with by several aut,hors (115, 120). Methods of removing impurities have been devised and perfect recovery of added thiamine is claimed. Collaborative results of t.he Association of Official Agricultural Chemists (108) indicate t.hat the fluorometric method for vitamin B, comparee favorahly with the microbiological method. This report shows that there is no statistically significant difference between the two. Klatzkin and his associates (38) in working with malted preparations have obtained much better agreement with the fluorometric method than .ivit,h the microbiological in the determination of riboflavin. Hoshino ( 7 7 ) and his associates give data for riboflavin determination in 40 foods and Engel and Hendricks (41) have determined it in cattle foods. The lumiflavin method for the determination of vitamin B2 has been used on various natural products (55, S1, 82, 92, 107, 136, 166). I n general, the lumiflavin which is formed by irradiation of riboflavin is extracted with chloroform and determined by the intensity of it's fluorescence rather than by extinction or colorimetric methods. The chemical determination of vitamin HI? by colorimetric and fluorometric methods is described by Boxer and Richards (18). The latter method employs t,he reaction with alloxan, forming highly fluorescent 6,7-dimethylalloxaaine. The composition of the fluorescent oxidation product of adreria-

line has been postulated by 1,und (104) and Ehrl6n (38). Lund has studied the determination of adrenaline extensively (103, loa. I n the fluorometric procedure adrenaline is oxidized with manganese dioxide to adrenochrome, which is converted by alkali to the highly fluorescent adrenolutine. Ascorbic acid will prevent the oxidation of adrenolutine by the air. By comparison of the fluorescence of sample with and without ascorbic acid present, the adrenolutine content can be determined. Annersten and and others (66) report excellent results for the associates (4, determination of adrenaline, in which sodium thiosulfate is used to prevent oxidation and, for comparison, the fluorescence of adrenaline is extinguished with iormaldehyde. The concentration of the alkali in this determination is important (48, 73). Iiatelson and others (117) have shown that adrenaline solution in aqueous ammonia containing a primary amine yields a fluorescent product which can be extracted with butyl alcohol. Paper chromatography has been applied to the determination of adrenaline by Shea (149). The chromatograph is developed by holding the paper in ammonia fumes. The fluorometric method for the deteimination of natural estrogens in various products has becn described by several authors (39, 4W, 67). The method of Garst and others (67) is based on the condensation reaction of phenolic steroids p i t h phthalic anhydride. Fierro del Rio and Arrieta Aupart (47) use antimony chloride as a reagent to give a green fluorescence w t h estrone and estradiol. The fluorescent spectra of nine natural estrogens after heating R ith sulfuric acid have been determined by Bates and Cohen (IO). Dh&-6 and Laszt (35) in an article on an absorption and fluorescence study of the Salkorvski reaction report data that are of interest in the determination of estrogens. hureomycin in buffered qolution can be determined by its yel]om fluorescence (99, 139) and the sensitivity can be increased by extraction Tyith n-butyl alcohol (140). Saltzman (137) claims that the accuracy of the fluorometric method for aureomycin is about the same as that of the microbiological method. The Banierjees (8) have reported on the use of the fluorometric method for estimating nicotinamide in cereals, legumes yeast, vegetables, and animal matter. The procedure avoids interfering vegetable components. Carpenter and Kodieck (63) give detailed directions for the determination of W-methylnicotinamide in urine and its differentiation from coenzyme 1. The fluorometric procedure for the determination of tryptophan has been the object of considerable study. This is based on the green fluorescence produced by tryptophan in perchloric acid. Indole is shov,n not to interfere and a linear relationship is obtained for as l o v as 1 to 8 micrograms of tryptophan (69). Of twenty amino acids examined only tryptophan, histidine, and citrulline exhibited a fluorescence (178). The technique for the stud> of nucleic acids by means of paper chromatography and ultraviolet photography is described. Goeller and Sherry (60) and Schou (146) have reported that pyridine nucleotides in the blood may be determined by the fluorescent compound v hich results when these materials are heated with alkali in acetone. Various inorganic ions have been found to have an extinction effect on the fluorescence of xanthopterin (168) and a quantitative study of the fluorescence spectra of this compound and folic acid has been made (31). The antihistaminics, which are 2substituted pyridine derivatives, form fluorescent compounds by means of a modified ryanogen bromide reaction which may be useful in their determination (124). A method of distinguishing ether-insoluble urinary porphyrins from uroporphyrin is based on the red fluorescence of these compounds in conjunction Fith adsorption and leaching procedures (20). The fluorometric determination of rotenone has been studied by Fonseca (60). Rotenone gives a blue fluorescence in alcohol, chloroform, acetone, and benzene, but is green in carbon tetrachloride. l h e chloroform extract seems best for the analysis. Pesez (165) has shown that gitoxin in phosphoric acid gives an intense bluish green fluorescence which may be used to deter-

V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 mine this substance. The fluorescence band of gitoxin under the conditions outlined is from 4600 to 5700 A. .4lkaloids of Rawolj a serpcntina ( 6 ) and of ergot (114) have been determined by fluorescence methods and the calabash curare alkaloids have been identified by their fluorescence after a paper chromatographic separation (144). I n the microdeterinination of belladonna, quinine sulfate serves as an excellent fluorescent acid-base indicator (91). The fluorometric method for the determination of quinine in oily niedicines is claimed by Jagle (80) to be more rapid, simple, and precise tha.n older methods. Several nat,ural products such as peanut oil and olcic acid hare been andyzed. The oil solution in ether is extracted by agitation with 0.1 N sulfuric acid. The fluorescence intensity of this sample is read in a fluorometer and t,hen it is treated witah concentrated hydrochloric acid and read again; thedillerence in readings is indicativeof thequininepresent. The assay of commercial quinine solutions also presents a prxctical use of fluorometric analysis (63). Edgar and Sokolow (37) have recorded their experiences with the fluorometric deterinination of quinidine in blood and reach th? conclusion that the method is good for clinical purposes. The ant,itrypanosome drug, ant,rycide, forms a highly fluorescent salt n i t h eosin which permits identification of the drug i n concentrations of 40 micrograms per lit,er of plasma with considerable accuracy. As no other tertiary base reacts with the dye, the method is specific (150). The determination of 3-hydroxyanthranilic acid in met,abolism studies may be accomplishpd by fluorometric means (17). The enzyme decomposition product of 3-hydroxynnthranolic acid is nonfluorescent. A new test for identifying bile acids has been devised by hIizuhara (111). The bile acid is dissolved in alkaline solution and potassium ferricyanide is added. The solution is extracted with butyl alcohol and the extract is examined under ult’raviolet light. The color of the fluoresceiice is characterist,ic of the position of t,he ketonic group. Qualit,ative examination of flours such as wheat,, corn, and soyhean under ultraviolet light is an import,ant, aid in clnssifying flours (86). The identification of 2-chloro-4-dimethylamino-6methylpyrimidine, Iyhich is sold as a rat poison, may be accomplished by observing t,he fluorescence after its reaction with resorcinol (64). Christensen and Latif (25) have used fluorometric methods successfully in the assay of senna leaves. h special fluora-illuminator has been designed by Schiller (14.2) for ohserving cutaneous fluorescence when it is used as an indicator of cnpillay? circulat,ion. Mose and his aesociates (115) have used a water extrac,t of thioflavine S for fluorescence visualization of the blood vessels. This reagent, is not toxic; it has a strong affinity for the blood vessel walls, and is strongly fluorescent. Fluorescent microscopy finds many applications in the identificat,ionof cert,ain biological materials. Kagner (167) uses thioflavin S as a stain for erythrocytes. Strugger (156) discusses the general procedure for staining live cells wit.h fluorescent dyes and gives 40 references. This same topic is the subject of articles by Ilofler (76) and Dangl (28). The possibility of the use of fluorescence in the study of lignin has been investigated by Kisser and Wittmann (88). The applications of fluorescence microscopy in food and drug malysis are reviewed by King arid Weston (87). Their paper includes a diagram of the ultraviolet illumination arrangement and a description of the techniques employed in the examination uf solid and liquid materials. LITERATURE CITED (1) Alford. W.C., and Daniel. J.H., ANAL.CHEM., 23, 1130 (1951).

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